Quantitative determination method for reactive sulfur

Abstract

Provided is a quantitative determination method for sulfur compounds in a target group selected from a plurality of groups, e.g., a group including cysteine and related reactive sulfurs, including the steps of: performing an LC/MS/MS measurement of a standard substance having a known concentration of a base compound, e.g., cysteine, in the target group; acquiring quantitative reference information used for quantitative determination of the base compound and other reactive sulfurs, based on the measurement result, assuming that the signal intensities of sulfur compounds in the same group show a predetermined relationship when their concentrations are equal; performing an LC/MS/MS measurement for each sulfur compound in a specimen, under an analysis condition determined beforehand so that the signal intensities of sulfur compounds in the same group show the predetermined relationship when their concentrations are equal; and determining the quantity of each sulfur compound, using the measurement result and quantitative reference information.

The invention claimed is: 
     
         1 . A quantitative determination method for a reactive sulfur, which is a method for determining a quantity of a sulfur compound including a reactive sulfur in a specimen by means of a chromatograph mass spectrometer, where the sulfur compound is included in at least one group as an analysis target selected from groups comprised of a group including cystine and reactive sulfurs related to cystine, a group including cysteine and reactive sulfurs related to cysteine, a group including oxidized glutathione and reactive sulfurs related to oxidized glutathione, and a group including reduced glutathione and reactive sulfurs related to reduced glutathione, the method comprising:
 a standard substance measurement step for performing a measurement by a chromatograph mass spectrometer, for a standard substance having a known concentration of a base compound which is cystine, cysteine, oxidized glutathione or reduced glutathione included in the group as the analysis target;   a quantitative reference information acquisition step for acquiring quantitative reference information used for determining quantities of the base compound and other reactive sulfurs included in the group as the analysis target, based on a measurement result obtained by the standard substance measurement step, on an assumption that signal intensities of a plurality of sulfur compounds included in the same group show a predetermined relationship when concentrations of the plurality of sulfur compounds are equal;   a specimen measurement step for performing a measurement of a specimen by a chromatograph mass spectrometer for each sulfur compound included in the group as the analysis target, where the measurement is performed under an analysis condition determined beforehand for each sulfur compound so that signal intensities of a plurality of sulfur compounds included in the same group show the predetermined relationship when the concentrations of the plurality of sulfur compounds are equal; and   a quantitative determination step for determining the quantity of each sulfur compound included in the group as the analysis target, using a measurement result obtained by the specimen measurement step and the quantitative reference information.   
     
     
         2 . The quantitative determination method for a reactive sulfur according to  claim 1 , wherein the quantitative determination step comprises determining a concentration ratio of a plurality of sulfur compounds included in each group. 
     
     
         3 . The quantitative determination method for a reactive sulfur according to  claim 1 , wherein the quantitative reference information acquisition step comprises determining a calibration curve which shows a relationship between concentration and signal intensity as the quantitative reference information, and the quantitative determination step comprises performing absolute quantitative determination by an external standard method using the quantitative reference information. 
     
     
         4 . The quantitative determination method for a reactive sulfur according to  claim 1 , wherein the chromatograph mass spectrometer is a liquid chromatograph-triple mass spectrometer, and the analysis condition to be tuned for each sulfur compound in the specimen measurement step includes multiple reaction monitoring transition, collision energy and mass-resolving power. 
     
     
         5 . A quantitative determination method for a reactive sulfur, which is a method for determining quantities of a plurality of sulfur compounds each of which includes a reactive sulfur in a specimen by means of a chromatograph mass spectrometer, where the plurality of sulfur compounds are included in at least one group as an analysis target selected from groups comprised of a group including cystine and reactive sulfurs related to cystine, a group including cysteine and reactive sulfurs related to cysteine, a group including oxidized glutathione and reactive sulfurs related to oxidized glutathione, a group including reduced glutathione and reactive sulfurs related to reduced glutathione, a group including sulfuric acid and inorganic sulfur compounds related to sulfuric acid, and a group including side-reaction products generated from sulfuric compounds by a derivatization treatment for stabilizing a reactive sulfur, the method comprising:
 a pretreatment step for performing, on a specimen, the derivatization treatment for stabilizing a reactive sulfur by using a predetermined derivatization reagent; 
 a specimen measurement step for performing a measurement of a specimen by a chromatograph mass spectrometer for each sulfur compound included in the group as the analysis target after a pretreatment of the specimen by the pretreatment step, where the measurement is performed under an analysis condition determined beforehand for each sulfur compound so that signal intensities of a plurality of sulfur compounds included in the same group show a predetermined relationship when concentrations of the plurality of sulfur compounds are equal; and 
 a quantitative determination step for performing relative quantitative determination of the plurality of sulfur compounds included in the group as the analysis target, based on a measurement result obtained by the specimen measurement step. 
 
     
     
         6 . The quantitative determination method for a reactive sulfur according to  claim 5 , wherein the analysis condition determined beforehand for each sulfur compound is determined so that the signal intensities of the plurality of sulfur compounds become substantially equal to each other. 
     
     
         7 . A quantitative determination method for a reactive sulfur, which is a method for determining a quantity of a sulfur compound including a reactive sulfur in a specimen by means of a chromatograph mass spectrometer, the method comprising:
 a standard substance measurement step for performing a measurement by a chromatograph mass spectrometer, for a standard substance having a known concentration of a sulfur compound included in a plurality of sulfur compounds forming one group which includes a target sulfur compound whose quantity is to be determined, where the plurality of sulfur compounds forming one group are identical in structure except for a chain combination of sulfur atoms and are different in a number of sulfur atoms forming the chain combination, and where the number of sulfur atoms forming the chain combination in the sulfur compound concerned is one or the smallest number in the group;   a quantitative reference information acquisition step for acquiring quantitative reference information used for determining quantities of the sulfur compounds included in the same group, based on a measurement result obtained by the standard substance measurement step, on an assumption that signal intensities of a plurality of sulfur compounds included in the same group show a predetermined relationship when concentrations of the plurality of sulfur compounds are equal;   a specimen measurement step for performing a measurement for the target sulfur compound in a specimen by a chromatograph mass spectrometer, where the measurement is performed under an analysis condition determined beforehand for each sulfur compound so that signal intensities of a plurality of sulfur compounds included in the same group show the predetermined relationship when the concentrations of the plurality of sulfur compounds are equal; and   a quantitative determination step for determining the quantity of the target sulfur compound, using a measurement result obtained by the specimen measurement step and the quantitative reference information.